Expression, Purification And Monoclone Antibody Preparation Of SARS Small Envelope Protein

Severe acute respiratory syndrome (SARS), also known as infectiveatypical pneumonia, is a new type of respiratory syndrome arose first in thesouthern China in the middle of November 2002. After that, the prevalencesoon erupts in the mainland, Hong Kong, Canada, Singapore and Vietnam. In abrief time of several months, it spreads 30 countries, 8,000 people wereinfluenced, and 1,000 people died of it. The prevalence of SARS not onlyseverely threatens the health and life of the human beings, but also has greatinfluence on the society and economy. The medical and scientific workers didthe best to meet the challenge of this unexpected calamity. After the globalalarm of SARS by the world health organization, the cause of SARS has beenidentified as a novel corona virus( or corona virus variant)-SARS-CoV bythe corporation of 13 top laboratories of 11 countries and regions within amonth. The preliminary genome remark of has been finished on the basis ofopen reading frames analysis through sequence similarity to the known coronaviruses. According to the gene coding, the anticipating protein includes RNApolymerase protein, the small envelope (E) protein, the spike (S) protein, thematrix (M) protein and the nucleocapsid (N) protein.The exploitations of SARS vaccine and diagnostic kit based on virusinactivation are great breakthroughs. But this technology need to extract virusfrom cultured cells, and can only be carried on in P3 stage lab. So it may havethe limitation of reproducibility and standardization. The methods based onantigen recombination can be more repetitive, easier to be controlled and hashigher biology security.The former researches of animal coronavirus structural protein find that Eprotein of coronavirus, which is the smallest, has significant effects in virusbudding with the M protein. Our research plan to express recombinant SARSE proteins with procaryon E coli system, achieve a great quantity of purifiedrecombinant antigen, and detect serum anti E protein antibody of SARSpatients. We also try to prepare the anti rat SARS-E protein monoclonalantibody.Our work includes the following parts:1. Obtain The SARS-E GeneWe synthesize the gene encoding the small envelope protein (E protein)according to the published SARS corona virus CUHK-W1 complete sequencewith DNA synthesizer of TaKaRa Company. We use this method to avoid theproblem of biological security of handling virus.2. Obtain The E Gene Fusion ProteinWe clone the SARS-E gene fragment on the expression vector;constructthe recombinant expression vector pET28a-E. The product is proven to becorrect according to the digestion restriction enzyme. Then transform E. coliBL21(DE3)with the recombinant plasmid, after the IPTG induction;weobtain the soluble highly expressed fusion protein. The result of SDS-PAGEelectrophoresis show that the molecular mass of the fusion protein is about27KD, agree with the anticipation.3. Western Blot Appraisement of Recombinant ProteinThe whole expressed bacteria do the SDS-PAGE electrophoresis ofbefore and after induction. Transfer the protein on nitrocellulose filter withtransferring electrophoresis;block it with confining liquid containing 10% calfserum. Then we use the convalescent serum of SARS patient as monoantigen,the HRP marked anti human goat IgG as diploantigen to react with the proteinseparately. The coloration solution were colored. The result showed that thefusion protein whose molecular weight is about 27KD can specially binding toSARS patients' serum. All confirm the expressed recombinant protein isSARS-E protein, and with well antigenicity.4. Obtain Abundant Recombinant Proteins With FermentationCultureIn order to get enough recombinant antigen for further research, we use30L fermentation tank to culture recombinant SARS-E protein in mass culture.We make the preliminary exploration to optimize the fermentation condition.We observed that the alteration of induction temperature, dissolved oxygenand IPTG concentration could influence the expression and solubility of theprotein. The results show that the OD600 can attain 10 when the proteincultured for 8h at 30℃ before induction. Induce the expression at 37℃ for 4h;control the dissolved oxygen not below 30%. The expression of the interestprotein is high and the expression product is soluble. When the concentrationof IPTG between 0.5-1.5mmol/L, the induction effect has no significantdeviation. We also try to use lactose to replace the IPTG induction, the effect issimilar to IPTG induction, it hint that lactose can be used in engineeringbacteria fermentation culture as a cheap, asepsis, and harmless inductor.5. Purifying the Recombinant ProteinWith deeper exploration, we suspense some fermentation culturedengineering bacteria in 20mmol/L Tris.Cl buffer at pH8.0, break the bacteriawith hypersound, and collect the supernatant with centrifugalization.First, the solution pass through the DEAE Sepharose FF negion column,under this condition, the objective protein directly flow out without sticking,and many hybridprotein can stick on it, so it can be purified preliminarily.Then the flow with the objective protein pass through the SP SepharoseFF powerful cathodolyte column, the protein can be eluted under 500mmol/LNaCl with NaCl gradient elution. Most hybridprotein has been removed;hydrophobic chromatography and molecular sieve were both not effective withthe hybridprotein that can not be removed with ion exchange chromatography.At last, we use the BLUE Sepharose CL-6B column to effectivelyseparate the objective protein and the hybridprotein with their difference ofnonspecific binding capability with the medium.The purity can above 95% with the SDS-PAGE running gel scananalysis.6. ELISA Detection of purifying recombinant SARS-E ProteinThe patients' serums were proved to be masculine with RT-PCR. Theserum of both patients and devotees were diluted in 1:50, the ratios of SARSpatients' A492 and the devotees' A492 is above 2.1, demonstrate the significantdifference between the two. It implicates that the expressed purifiedrecombinant SARS-E protein can be used in SARS patients' serologicaldiagnosis.7. Preparing the monoclonal antibodyWe immune the Balb/c mouse with purified recombinant E protein, theresult of sepharose double diffusion experiment indicate that the fusion proteincan stimulate the body to produce corresponding antibody. After that, we usehybridoma fusion method to successfully prepare a mouse monoclonalantibody after 5 cloning screening. The result of Western blot analysisdemonstrates that the prepared monoclonal antibody can specially react withSARS-E protein, the controlled SARS-S2 protein not have this reaction. Itproves that the screened monoclonal antibody is anti SARS-E proteinmonoclonal antibody.Our research prepares abundant recombinant SARS-E protein antigen,and this antigen has specialized reaction with SARS patients' serum onconvalescent period and has significant reference on the research of SARSdiagnostic kit. The prepared monoclonal antibody can provide basis for furtherresearch of the characteristics and function of nucleocapsid protein and itsantibody. Preparing human anti SARS monoclonal drug can also benefit fromit.